CN109402085B

CN109402085B - Recombinant strains, methods for synthesizing simvastatin, and related enzymes

Info

Publication number: CN109402085B
Application number: CN201810112817.6A
Authority: CN
Inventors: 吕雪峰; 梁波; 杨勇; 黄雪年; 郑玲辉; 滕云
Original assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2018-02-05
Filing date: 2018-02-05
Publication date: 2021-02-12
Anticipated expiration: 2038-02-05
Also published as: CN109402085A

Abstract

The invention belongs to the technical field of biotechnology and medicine, and provides a recombinant strain and a method for synthesizing simvastatin, and related enzymes, wherein the recombinant strain expresses 2-methylbutyrate side chain hydrolase and acyltransferase simultaneously. The simvastatin synthesis method disclosed by the invention is simplified in steps, low in impurity content and mild in reaction conditions.

Description

Recombinant strains, methods for synthesizing simvastatin, and related enzymes

Technical Field

The invention belongs to the technical field of biotechnology and medicine, and relates to synthesis of simvastatin.

Background

Cardiovascular and cerebrovascular diseases have become the first killers of human beings, and the incidence rate is increased year by year. Hyperlipidemia is one of the main causes of cardiovascular and cerebrovascular diseases, so that the risk of cardiovascular and cerebrovascular diseases can be effectively reduced by reducing the blood lipid. Statins are the most commonly used of the lipid-regulating drugs currently used in clinical practice. Lovastatin was isolated from the metabolites of aspergillus terreus by the scientist of merck in 1979 and approved by the FDA in the united states in 1987 as the first statin approved for marketing in the world. Then, the company synthesizes simvastatin by taking lovastatin as a raw material, the alpha-carbon atom of the side chain of the C-8 butyric ester of the simvastatin is one more methyl than that of the lovastatin, and the action mechanisms of the lovastatin and the lovastatin are the same. In recent years, clinical studies have shown that atorvastatin and then simvastatin are the best at lowering low density lipoprotein cholesterol, and therefore, the sales of both are also listed on the leaderboard. The process and cost for producing simvastatin have become important factors for the market competition of the product.

Currently, the production process of simvastatin is mainly a chemical synthesis method, as described in CN1290261A, CN1612872A and CN1493570A, and the basic production process consists of two steps of reaction, wherein the first step is to generate monacolin J from lovastatin, and the second step is to generate simvastatin from monacolin J. These processes have the disadvantages of long conversion times, harsh reaction conditions, high environmental pollution, and the need for expensive and hazardous chemical reagents. With the development of biotechnology, the biosynthesis of simvastatin is receiving more and more attention. Among them, the biotransformation technique of the second reaction has been reported, which utilizes acyltransferase, which can transfer the acyl group of acyl thioester such as α -dimethylbutyryl mercaptopropionic acid methyl ester (DMB-S-MMP) to the hydroxyl group at C8 of monacolin J to synthesize simvastatin as described in CN102703539A, CN103725726A, CN102695792A, CN102574896A, CN101490271, CN 102712678A. However, the biotransformation technique of the first step reaction has not been established so far. The relevant literature reports a 2-methylbutyryl side chain hydrolase (PcEST) which can specifically hydrolyze lovastatin to form monacolin J, and by introducing the PcEST into a lovastatin-producing strain, a monacolin J-producing Aspergillus terreus strain is obtained with a hydrolysis efficiency of more than 90% (Huang XN, Liang YJ, Yang Y, Lu XF. (2017), Single-step process of the simvastatin refractory monacolin J by engineering of an industrial strain of Aspergillus terreus, plant. Eng.,42, 109-114).

However, this approach only improves the first reaction step, and the second reaction step, which is carried out to obtain simvastatin, is relatively cumbersome.

Disclosure of Invention

The invention mainly aims to develop a high-efficiency, low-cost, green and environment-friendly simvastatin total biosynthesis process taking lovastatin and acyl thioester as substrates by simultaneously producing 2-methylbutyryl side chain hydrolase and acyltransferase in a microorganism strain.

In a first aspect, the present invention provides a 2-methylbutyrate side chain hydrolase which is a mutant obtained by site-directed mutagenesis of a wild-type PcEST (SEQ ID NO:1), the site of mutagenesis including 106, 140 and 304, and the forms of mutagenesis are D106G, Q140L and S304G, and therefore, the sequence of the 2-methylbutyrate side chain hydrolase is:

MDTTFQAAIDTGKINGAVVCATDAQGHFVYNKATGERTLLSGEKQPQQLDDVLYLASATKLITTIAALQCVEDGLLSLDGDLSSIAPELAAKYVLTGFTDDESPLX₁₀₆DPPARPITLKMLLTHSSGTSYHFLDPSIAKWRAX₁₄₀YANPENEKPRLVEEMFTYPLSFQPGTGWMYGPGLDWAGRVVERVTGGTLMEFMQKRIFDPLGITDSQFYPVTREDLRARLVDLNPSDPGALGSAVIGGGGEMNLRGRGAFGGHGLFLTGLDFVKILRSLLANDGMLLKPAAVDNMFQQHLGPEAAASHRAALAX₃₀₄PLGPFFRVGTDPETKVGYGLGGLLTLEDVDGWYGERTLTWGGGLTLTWFIDRKNNLCGVGAIQAVLPVDGDLMADLKQTFRHDIYRKYSAWKGQQ, wherein X₁₀₆Is D or G, X₁₄₀Is Q or L, X₃₀₄Is S or G, and the sequence is not the sequence shown in SEQ ID NO. 1.

Specifically, the 2-methylbutyrate side chain hydrolase is respectively:

(1) PcEST/D106G: namely, the 106 th site of the wild type PcEST amino acid sequence is mutated from D to G; the amino acid sequence is shown as SEQ ID NO. 3;

(2) PcEST/Q140L: namely, the 140 th position of the wild type PcEST amino acid sequence is mutated from Q to L; the amino acid sequence is shown as SEQ ID NO. 4;

(3) PcEST/S304G: namely, the 304 th site of the wild type PcEST amino acid sequence is mutated from S to G; the amino acid sequence is shown as SEQ ID NO. 5;

(4) PcEST/D106G/Q140L: namely, the 106 th position of the wild type PcEST amino acid sequence is mutated from D to G, and meanwhile, the 140 th position is mutated from Q to L; the amino acid sequence is shown as SEQ ID NO. 6;

(5) PcEST/D106G/S304G: namely, the 106 th position of the wild type PcEST amino acid sequence is mutated from D to G, and simultaneously, the 304 th position is mutated from S to G; the amino acid sequence is shown as SEQ ID NO. 7;

(6) PcEST/Q140L/S304G: namely, the 140 th position of the wild type PcEST amino acid sequence is mutated from Q to L, and simultaneously, the 304 th position is mutated from S to G; the amino acid sequence is shown as SEQ ID NO. 8; or

(7) PcEST/D106G/Q140L/S304G: namely, the 106 th position of the wild type PcEST amino acid sequence is mutated from D to G, meanwhile, the 140 th position is mutated from Q to L, and meanwhile, the 304 th position is mutated from S to G; the amino acid sequence is shown in SEQ ID NO. 9.

In this document, the abbreviation of PcEST/D106G as D106G indicates protein mutants, unless otherwise specified. The skilled person can clearly distinguish whether D106G represents a mutant form or mutant, depending on the context. Other protein mutants are represented in the same manner.

In a second aspect, there is provided a biological material related to the 2-methylbutyrate side chain hydrolase of the invention, which is:

(1) a nucleic acid molecule encoding a 2-methylbutyrate side chain hydrolase according to the invention; preferably, the amino acid sequence of the 2-methylbutyrate side chain hydrolase is shown as SEQ ID NO 3-9; further preferably, the nucleic acid molecule is PcEST/D106G (SEQ ID NO:12), PcEST/Q140L (SEQ ID NO:13), PcEST/S304G (SEQ ID NO:14), PcEST/D106G/Q140L (SEQ ID NO:15), PcEST/D106G/S304G (SEQ ID NO:16), PcEST/Q140L/S304G (SEQ ID NO:17), PcEST/D106G/Q140L/S304G (SEQ ID NO: 18); or

(2) An expression cassette, a recombinant vector, a recombinant cell or a recombinant microorganism comprising the nucleic acid molecule of (1).

With respect to the above-mentioned biomaterials, the sequence of a nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase can be easily known by those skilled in the art from the amino acid sequence of the enzyme provided, and an expression cassette, a recombinant vector, a recombinant cell and a recombinant microorganism comprising the nucleic acid molecule to express a protein can be easily selected, prepared and used.

In a third aspect, the invention provides the use of the 2-methylbutyric acid side chain hydrolase of the invention for hydrolyzing statins containing 2-methylbutyric acid side chains; preferably, the statin substance containing a 2-methylbutyric acid side chain is lovastatin, mevastatin or pravastatin; further preferably, the statin is in acid, lactone or salt form.

In a fourth aspect, the present invention provides a recombinant strain for synthesizing simvastatin, which expresses both the 2-methylbutyrate side chain hydrolase of the present invention and the acyltransferase.

Preferably, the recombinant strain is escherichia coli; further preferred is the strain Escherichia coli BL21(DE 3).

More preferably, the amino acid sequence of the 2-methylbutyrate side chain hydrolase is shown in any one of SEQ ID NO 3-9, and the amino acid sequence of the acyltransferase is shown in SEQ ID NO 2.

Further preferably, the recombinant strain carries a gene encoding 2-methylbutyrate side chain hydrolase and a gene encoding acyltransferase.

Further preferably, the nucleotide sequence of the coding gene of the 2-methylbutyrate side chain hydrolase is shown in any one of SEQ ID NO 12-18, and the nucleotide sequence of the coding gene of the acyltransferase is shown in SEQ ID NO 11.

In a fifth aspect, there is provided a method of constructing a recombinant strain of the invention, comprising the steps of: the plasmid carrying the coding gene of the 2-methylbutyrate side chain hydrolase and the plasmid carrying the coding gene of the acyltransferase are co-transformed into escherichia coli, and a recombinant strain simultaneously expressing the 2-methylbutyrate side chain hydrolase and the acyltransferase is obtained by screening.

Methods for plasmid cotransformation are well known in the art. The skilled person can select suitable plasmids and co-transformation methods and screening methods according to the description to finally obtain the desired recombinant strain.

Preferably, the nucleotide sequence of the coding gene of the 2-methylbutyrate side chain hydrolase is shown in any one of SEQ ID NO 12-18, and the nucleotide sequence of the coding gene of the acyltransferase is shown in SEQ ID NO 11.

In a sixth aspect, the invention provides an application of the recombinant strain or its bacterial suspension or its fermentation broth or its bacterial powder in synthesizing simvastatin.

Specifically, the recombinant strain can synthesize a product simvastatin by taking lovastatin and acyl thioester as substrates: adding lovastatin and acyl thioester into a solution containing a whole cell catalyst of an escherichia coli recombinant strain, enabling the lovastatin to enter cells, hydrolyzing by 2-methylbutyrate side chain hydrolase to generate monacolin J, synthesizing a simvastatin product by the monacolin J and the acyl thioester entering the cells under the action of acyltransferase, and transporting the simvastatin out of cells.

Therefore, in a seventh aspect, the invention provides a method for synthesizing simvastatin, which comprises using the recombinant strain of the invention, and taking lovastatin and acyl thio ester as substrates, and using escherichia coli whole cell to biologically catalyze the transformation of lovastatin to synthesize simvastatin. Specifically, the strain is cultured to express 2-methylbutyrate side chain hydrolase and acyltransferase, and then the strain is freeze-dried to obtain strain powder; adding the above powder into solution containing lovastatin (15mM) and acyl thioester (20mM) to make final concentration 3-6mg/ml, and reacting at 25 deg.C and 200rpm for 1-5 h. The concentrations of simvastatin, lovastatin and monacolin J are detected by high performance liquid chromatography, and the highest conversion rate of the lovastatin into simvastatin reaches 92%.

Compared with the prior art, the method takes lovastatin and acyl thioester as raw materials, simvastatin is generated by the action of intracellular hydrolase and acyltransferase, not only the reaction steps are greatly reduced, but also the transformation rate of lovastatin synthesized simvastatin is improved from about 50 percent to over 90 percent by optimizing recombinant strains, the content of impurities in simvastatin products is reduced, and particularly the residue of lovastatin is below 0.1 percent. Therefore, the method provided by the invention is simple and easy to operate, mild in reaction condition, safe, green and environment-friendly.

Drawings

FIG. 1: and (3) a nucleic acid electrophoresis picture of the wild-type gene which is obtained by PCR amplification and codes the 2-methylbutyryl side chain hydrolase PcEST. M is a DNA molecular weight standard. The template for PCR amplification was the pEASY-E2-PcEST plasmid (Huang XN, Liang YJ, Yang Y, Lu XF. (2017). Single-step production of the simvastatin monoclinic J by engineering of an industrial strain of Aspergillus terreus, Metal. Eng.,42, 109-114).

FIG. 2: and (3) carrying out electrophoresis picture on the nucleic acid of the gene encoding the acyltransferase LovD9 obtained by PCR amplification. M, DNA molecular weight standard. The template for PCR amplification was the coding gene (codon optimized according to the codon preference of E.coli) carrying LovD9(PBD ID:4LCM) synthesized in whole gene.

FIG. 3: electropherograms of PcEST and LovD9 protein expression in recombinant strains. M is a protein molecular weight standard. The PL1 strain was a recombinant strain of E.coli encoding both LovD9 and the wild type PcEST, and the PLM3 strain was a recombinant strain of E.coli encoding both LovD9 and PcEST mutant D106G/Q140L/S304G. The red arrow indicates the LovD9 protein (46kDa) and the black arrow indicates the PcEST protein (43 kDa). S is the cell disruption supernatant, i.e., the soluble fraction. I is the cell disruption pellet, i.e. the insoluble fraction.

FIG. 4: high performance liquid chromatogram of recombinant strain (the thallus concentration is 4.38mg/ml) for synthesizing simvastatin by taking lovastatin and acyl thioester as substrates. The PL1 strain was a recombinant strain of E.coli encoding both LovD9 and the wild type PcEST, and the PLM3 strain was a recombinant strain of E.coli encoding both LovD9 and PcEST mutant D106G/Q140L/S304G. The retention times of monacolin J, lovastatin and simvastatin were 2.7min, 9.5min and 13.4min, respectively.

FIG. 5: the concentration of simvastatin synthesized by different concentrations of the strain is plotted as a function of time. The PL1 strain was a recombinant strain of E.coli encoding both LovD9 and the wild type PcEST, and the PLM3 strain was a recombinant strain of E.coli encoding both LovD9 and PcEST mutant D106G/Q140L/S304G.

FIG. 6: (A) is an electrophoretic analysis chart of the purification results of the PcEST wild type and mutant proteins; (B) is a graph of the results of enzyme activity measurements, with the unit of enzyme activity being defined as the amount of enzyme required to catalyze the production of 1 micromole of monacolin J per minute as one unit of activity (U). The ordinate in the figure indicates the number of enzyme activity units contained per mg of enzyme protein. WT: wild type PcEST.

FIG. 7: michaelis equation plot for PcEST wild type and mutant enzyme proteins, WT: wild type PcEST.

FIG. 8: electrophoretic analysis of soluble expression of PcEST wild-type and mutant proteins, WT: wild type PcEST. S: a soluble protein moiety; i: an insoluble protein fraction.

FIG. 9: thermostability assay pattern of PcEST wild type and mutant proteins, WT: wild type PcEST.

Detailed Description

The present invention will be described in further detail with reference to specific examples. These examples show detailed embodiments and specific procedures to aid understanding of the present invention, but the scope of the present invention is not limited to the following examples.

Unless otherwise specified, the reagents and instruments used in the following examples are those conventional in the art and are commercially available; the method used is also a routine method, and a person skilled in the art can clearly know how to complete the experiment and obtain corresponding results according to the content of the specification.

Example 1 construction of recombinant strains of Escherichia coli for the biocatalytic transformation of lovastatin synthetic simvastatin.

1. Construction of expression vector pRSFDuet-PcEST carrying wild-type 2-methylbutyryl side chain hydrolase PcEST coding Gene

The coding gene of PcEST is obtained by conventional PCR amplification with primer 1 and primer 2 as primers (synthesized by Beijing Liu-Hua Dagen Co., Ltd.), and the primer sequences are as follows:

primer 1:5 'GGAATTCCATATGGATACCACCTTTCAGGCG 3'

Primer 2: 5 'CCGCTCGAGTCACTGCTGACCTTTCCAGGC 3'

The template was the pEASY-E2-PcEST plasmid (Huang XN, Liang YJ, Yang Y, Lu XF. (2017). Single-step production of the simvastatin precarsor monoacolin J by engineering of an industrial strain of Aspergillus terreus. Metal. Eng.,42, 109-114).

The PCR product was purified and recovered, and then subjected to nucleic acid electrophoresis detection, the result of which is shown in FIG. 1, and the size of the DNA fragment was 1200 bp. The cohesive ends were excised with restriction enzymes Nde I and Xho I, and ligated with the vector pRSFDuet-1 (available from Novagen) obtained by the same digestion using T4 ligase to obtain a recombinant vector pRSFDuet-pcest. The plasmid was transformed into competent cells of E.coli DH 5. alpha. and transformants were picked on LB plates containing kanamycin (final concentration 50. mu.g/ml), sequenced and the correct strain and the corresponding vector pRSFDuet-PcEST contained therein were screened as shown in the nucleotide sequence SEQ ID NO:10 of the PceST wild type.

2. Construction of expression vector pET22b-LovD carrying gene encoding acyltransferase LovD9

The encoding gene of LovD9(PBD ID:4LCM) was synthesized by a whole-gene synthesis method (synthesized by Beijing Liuhua DageneCo., Ltd., codon optimization was performed according to the codon preference of E.coli). The plasmid is used as a template, a primer 3 and a primer 4 are used as primers (synthesized by Beijing Liuhe Huada Gene Co., Ltd.), and the encoding gene of LovD9 is obtained by conventional PCR amplification, wherein the primer sequence is as follows:

primer 3: 5 'GGAATTCCATATGGGCAGCAACATTGATGCGGCTGTGGC 3'

Primer 4: 5 'CCCAAGCTTTTAGCCCTGCTGATACTGCGCATAAATCG 3'

The PCR product was purified and recovered, and then subjected to nucleic acid electrophoresis, and the result is shown in FIG. 2, in which the DNA fragment size was 1239 bp. The cohesive ends were excised with restriction enzymes Nde I and Hind III, and ligated with the vector pET22b (stored in the laboratory, and the conventional pET22b vector was modified to remove the signal peptide sequence) obtained by the same digestion using T4 ligase to obtain the recombinant vector pET22 b-lovd. The plasmid was transformed into competent cells of E.coli DH 5. alpha. and transformants were picked on LB plates containing ampicillin (final concentration 100. mu.g/ml), sequenced and the correct strain was selected according to the form shown in the nucleotide sequence SEQ ID NO:11 of LovD9 and the corresponding vector pET22b-LovD contained therein.

3. Construction of recombinant Escherichia coli containing PcEST and LovD9 coding genes

The pRSFDuet-pcest and pET22b-lovd vectors were co-transformed into competent cells of the E.coli expression strain BL21(DE3) in a molar ratio of 1: 1. Transformants were picked on LB plates containing both ampicillin (final concentration 100. mu.g/ml) and kanamycin (final concentration 50. mu.g/ml), sequenced, and the correct strains and the corresponding vectors pRSFDuet-PcEST and pET22b-LovD contained therein, i.e., recombinant E.coli carrying genes encoding PcEST and LovD9, were selected as the nucleotide sequences of PcEST wild type (shown as SEQ ID NO: 10) and LovD9 (shown as SEQ ID NO: 11), and named PL1 strain.

4. Expression of PcEST and LovD9 proteins

PL1 strain was cultured in 100ml at 37 ℃ to logarithmic growth phase, and then isopropyl-. beta. -D-thiogalactoside (IPTG, final concentration 0.1mM) as an inducer was added and cultured at 25 ℃ for 16 hours to induce protein expression. The cells were collected, suspended in 100ml of Tris-HCl buffer (50mM, pH8.0), sonicated, centrifuged, and the supernatant and pellet were separated, and the pellet was suspended in 100ml of Tris-HCl buffer (50mM, pH8.0), and 10. mu.l of the supernatant and pellet fractions were analyzed by SDS-PAGE, and the results are shown in FIG. 3. Both proteins PcEST and LovD9 were expressed in cells, but the soluble expression level of PcEST was low, indicating that PcEST wild type is easy to form inclusion body when expressed at 25 ℃, i.e. the protein is not folded correctly.

5. Determination of PcEST and LovD9 enzyme Activity

The PL1 strain was cultured in 100ml at 37 ℃ to the logarithmic growth phase, followed by addition of IPTG (final concentration of 0.1mM) as an inducer and culture at 25 ℃ for 16 hours to induce protein expression. The cells were collected and suspended in 100ml of Tris-HCl buffer (50mM, pH 8.0).

Adding newly prepared Tris-HCl buffer (50mM, pH8.0) and lovastatin into the above Tris-HCl buffer containing the cells to make the final concentration of cells OD₆₀₀1.0, final lovastatin concentration 0.5 mM. Reacting at 25 deg.C and 200rpm for 10min, centrifuging to remove thallus, adding equal amount of methanol into supernatant, and performing High Performance Liquid Chromatography (HPLC) (Agilent as chromatographic column)ZORBAX XDB-C18 (4.6X 150mm,5 μ M), acetonitrile, 0.1% phosphoric acid (50:50, v: v) at a flow rate of 1ml/min, 10 μ l of sample injection, and the enzyme activity of PcEST calculated as 2.54 + -0.07U/ml (enzyme activity unit U is defined as the amount of enzyme required to catalyze the production of 1 μ M of Monacolin J per minute), the lower activity of PcEST is due to the lower soluble expression level of PcEST protein (as shown in FIG. 3).

Adding newly prepared Tris-HCl buffer (50mM, pH8.0), monacolin J and alpha-dimethylbutyryl-S-methylmercaptopropionate (DMB-S-MMP) to the above Tris-HCl buffer containing the cells to make the final concentration of the cells OD₆₀₀1.0, final concentration of monacolin J is 0.5mM and final concentration of DMB-S-MMP is 2 mM. Reacting at 25 ℃ and 200rpm for 10min, centrifuging to remove thalli, adding methanol with the same amount into supernate, detecting generated simvastatin by using HPLC, and calculating to obtain the enzyme activity of LovD9 to be 9.55 +/-0.56U/ml (the unit U of the enzyme activity is defined as the enzyme amount required for catalyzing and generating 1 mu M simvastatin per minute).

Example 2 application of recombinant Escherichia coli strain PL1 in whole cell biocatalytic conversion of lovastatin to synthesize simvastatin

1. Preparation of fungal powder

PL1 strain was cultured in 1L at 37 ℃ to logarithmic growth phase, and IPTG (final concentration of 0.1mM) was added as an inducer, and the strain was cultured at 25 ℃ for 16 hours to induce protein expression. The cells were collected by centrifugation, washed twice with a volume of twice the volume of PBS buffer (50mM, pH8.0), pre-frozen overnight at-80 ℃ and freeze-dried for 24 hours to give a bacterial powder.

2. Production of simvastatin

0.5g, 0.7g and 1.0g of PL1 bacterial powder were taken, 160ml of Tris-HCl buffer (50mM, pH8.0) containing lovastatin (final concentration: 15mM) and methyl α -dimethylbutyrylthiopropionate (DMB-S-MMP) (final concentration: 20mM) was added to give final concentrations of 3.13mg/ml, 4.38mg/ml and 6.25mg/ml, respectively, mixed and divided into 9 equal portions, and reacted at 25 ℃ and 200 rpm. Samples were taken at regular intervals. Separating thallus and supernatant, adding equal volume of ethyl acetate into supernatant, mixing, evaporating by a rotary evaporator, and adding methanol to dissolve samples. Adding dichloromethane/methanol mixed solution (1:1, v: v) into the thallus, performing ultrasonic extraction, filtering to remove insoluble substances, evaporating the liquid in a rotary evaporator, and adding methanol to dissolve. And combining the supernatant with methanol solution of the thallus part, blowing the mixture with nitrogen, adding methanol with the same volume, and measuring the generated simvastatin by using HPLC. As can be seen from FIGS. 4 and 5, as the cell concentration increased, the time taken until the conversion rate reached the maximum decreased, specifically: when the concentration of the thalli is 3.13mg/ml, the conversion rate reaches the highest value after 1 hour, namely 49 percent; when the concentration of the thalli is 4.38mg/ml, the conversion rate reaches the highest value after 3 hours, namely 51.5 percent; when the cell concentration was 6.25mg/ml, the conversion rate reached the highest value, namely 53%, after 5 hours.

The reason for the lower conversion rate is that the PcEST wild-type in the PL1 strain has a low soluble expression level when expressed at around 25 ℃ (as shown in fig. 3), so that the activity of the whole-cell PcEST enzyme is low and significantly lower than that of LovD9, and therefore the lovastatin hydrolysis reaction catalyzed by the wild-type PcEST enzyme is the rate-limiting step in the whole simvastatin synthesis pathway, so that the total conversion rate is low. In addition, the inventor finds that the enzyme activity is reduced by 87% after the wild type PcEST is placed at 37 ℃ for 1 hour in earlier research, which indicates that the enzyme has poor thermal stability and the production cost caused by artificially controlling low temperature is greatly increased in industrial production. Therefore, the application value of the enzyme can be further improved by improving the soluble expression level of the enzyme and improving the performances such as thermostability, activity and the like.

Example 3 optimization and improvement of recombinant strains of E.coli for transformation of lovastatin synthetic simvastatin

1. Obtaining of mutant of 2-methylbutyrate side chain hydrolase

(1) Construction of wild type 2-methylbutyrate side chain hydrolase expression vector:

the gene coding the 2-methylbutyrate side chain hydrolase is obtained by conventional PCR amplification with the primer 1 and the primer 2 as primers (synthesized by Beijing Liuhua DageneCo., Ltd.). The primer sequences are as follows:

primer 1:5 'GGAATTCCATATGGATACCACCTTTCAGGCG 3'

Primer 2: 5 'CCGCTCGAGCTGCTGACCTTTCCAGGC 3'.

After the PCR product is purified, restriction enzymes Nde I and Xho I are used for enzyme digestion, the PCR product is connected with a pET28a-smt3 vector which is subjected to the same enzyme digestion, the obtained vector is named as pET-PcEST, plasmid transformation is carried out on escherichia coli BL21(DE3) competent cells, transformants are picked and sequenced, and correct strains and the corresponding vector pET-PcEST contained in the strains are screened according to the form shown by a PcEST wild type nucleotide sequence SEQ ID NO: 10.

(2) Construction of 2-methylbutyrate side chain hydrolase mutant:

A. construction of Single Point mutants (D106G, Q140L, S304G)

Mutation PCR: site-directed mutagenesis was performed using the QuikChange method. pET-pcest plasmid DNA is used as a template, primers (Table 1) are designed and synthesized according to mutation sites, and PCR amplification is carried out to obtain the gene for coding the 2-methylbutyrate side chain hydrolase mutant.

The PCR reaction system comprises 32.5 mu L of sterile water, 1 mu L of template DNA and 5 XPrimeSTAR HS buffer (Mg)²⁺plus), 4. mu.L of dNTP mixture (2.5 mmol/L each), 1. mu.L of forward primer (20 pmol/. mu.L), 1. mu.L of reverse primer (20 pmol/. mu.L), and 0.5. mu.L of TaKaRa PrimeSTAR HS polymerase. The reaction condition is pre-denaturation at 95 ℃ for 5 min; 10s at 94 ℃, 15s at 60 ℃ and 7.5min at 72 ℃ for 18 cycles; finally, extension is carried out for 10min at 72 ℃.

Enzyme digestion: the PCR product after the reaction was taken out, the Dpn I enzyme was added at a ratio of 1:50(Dpn I enzyme: PCR product, v: v), and the mixture was digested at 37 ℃ for 1 hour.

(iii) removed, transformed into competent cells of Escherichia coli BL21(DE3), spread on LB plates containing kanamycin (final concentration 50. mu.g/ml), and cultured overnight at 37 ℃.

After transformants grew, single clones were picked for sequencing.

B. The construction of the double-point mutant is that on the basis of the single-point mutant (such as D106G), primers of other single mutation points (such as Q140L forward direction and Q140L reverse direction (Table 1)) are used for carrying out PCR reaction, and mutation sites of other single-point mutations (such as Q140L) are introduced.

C. The three-point mutant is constructed by carrying out PCR reaction on the basis of a double-point mutant D106G/Q140L by using a primer S304G in the forward direction and a primer S304G in the reverse direction (Table 1) and introducing a mutation site of S304G.

TABLE 1 PCR primers used in site-directed mutagenesis methods

Primer and method for producing the same	Sequence (5 '→ 3')
		D106G Forward direction	ACGAAAGTCCGCTGGGCGATCCGCCGGCAC
D106G reverse direction	GTGCCGGCGGATCGCCCAGCGGACTTTCGT
		Q140L Forward	CAAAATGGCGCGCACTATACGCGAATCCGG
Q140L reverse direction	CCGGATTCGCGTATAGTGCGCGCCATTTTG
		S304G forward direction	CGCGCAGCACTGGCAGGTCCGCTGGGTCCG
S304G reverse	CGGACCCAGCGGACCTGCCAGTGCTGCGCG

And screening correctly mutated strains and corresponding vectors contained in the strains according to the mutation forms shown by the nucleotide sequences SEQ ID NO 12-18 of the single-point mutants, and carrying out subsequent experiments.

(3) Purification of 2-methylbutyrate side chain hydrolase wild-type and mutant proteins

Coli BL21(DE3) expressing the wild-type enzyme and the mutant enzyme was cultured at 37 ℃ to the logarithmic growth phase, IPTG (final concentration of 0.2mM) was added thereto, overnight induction expression was carried out at 16 ℃, the cells were collected and resuspended in 40ml of Tris-HCl buffer (20mM, pH8.0), and the cells were disrupted by sonication to obtain a crude enzyme solution which was purified by Ni-NTA. The protein was collected, digested overnight with Ulp1(Ulp1: PcEST mass ratio of 1:50), and purified by hanging Ni column back, most of the target protein passed through Ni column, while His-sumo bound to Ni column tightly, and needed high concentration (250mM) of imidazole to elute, so that purified protein could be obtained (FIG. 6A).

The sequence of each mutant enzyme was determined to be identical to that expected.

2. Characterization of 2-methylbutyrate side chain hydrolase mutants

(1) Determination of 2-methylbutyrate side chain hydrolase mutant enzyme Activity

The above prepared protein was taken for enzyme activity assay. The concentration of the enzyme protein was 0.05. mu.M, the concentration of the substrate lovastatin was 400. mu.M, the reaction was carried out at 37 ℃ for 10 minutes, and the produced monacolin J was measured by HPLC. The column used for the HPLC reaction was Agilent ZORBAX XDB-C18 (4.6X 150mm,5 μm), the mobile phase was acetonitrile: 0.1% phosphoric acid (50:50, v: v) at a flow rate of 1ml/min, and 10. mu.l of sample was introduced. As shown in FIG. 6B, the enzyme activities of the 2-methylbutyrate side chain hydrolases of mutants D106G, Q140L, S304G, D106G/Q140L and D106G/Q140L/S304G were 2.90 times, 2.51 times, 1.91 times, 5.10 times and 6.46 times, respectively, that of the wild type.

(2) Determination of kinetic parameters of enzymatic reactions of wild type and mutant of 2-methylbutyrate side chain hydrolase

The initial rates of the enzyme reactions under different conditions were determined at the following concentrations of enzyme and substrate lovastatin, and the curves of the Michaelis equation were plotted. As shown in fig. 7.

Enzyme concentration: wild type: 0.05 μ M; mutant (D106G, D106G/Q140L and D106G/Q140L/S304G): 0.02. mu.M.

Concentration of substrate lovastatin: wild type: 15 μ M, 25 μ M, 40 μ M, 50 μ M, 100 μ M, 200 μ M, and 300 μ M; mutant D106G: 15. mu.M, 20. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 400. mu.M and 500. mu.M; mutant D106G/Q140L: 15 μ M, 20 μ M, 25 μ M, 50 μ M, 100 μ M, 200 μ M, 250 μ M, 400 μ M, 500 μ M, and 600 μ M; mutant D106G/Q140L/S304G: 15 μ M, 20 μ M, 25 μ M, 50 μ M, 100 μ M, 200 μ M, 300 μ M, 400 μ M and 500 μ M.

The kinetic parameters of the enzymatic reaction are shown in table 2. kcat/K of mutants D106G, D106G/Q140L, D106G/Q140L/S304G_MCompared with wild type, the wild type gene has the advantages of 1.54, 4.0 and 5.06 times higher activity.

TABLE 2 kinetic parameters of the reaction of the 2-methylbutyrate side chain hydrolase wild-type and mutant

(3) Determination of soluble expression of 2-methylbutyrate side chain hydrolase wild-type and mutant proteins

100ml of Escherichia coli BL21(DE3) encoding wild-type and mutant 2-methylbutyrate side chain hydrolase was cultured at 37 ℃ to the logarithmic growth phase, IPTG (final concentration of 0.2mM) was added to induce expression of the protein at 25 ℃ and the bacterial solution was collected overnight, centrifuged after ultrasonication, the supernatant and the precipitate were separated, and the precipitate was suspended in the same volume of Tris-HCl buffer (50mM, pH 8.0). The same amount of supernatant and precipitated protein solutions were subjected to SDS-PAGE protein electrophoresis analysis, and the results are shown in FIG. 8. The majority of wild type protein is in the precipitation part, while the majority of proteins of mutant D106G, D106G/Q140L and D106G/Q140L/S304G are in the supernatant part, which shows that the soluble expression level of mutant D106G protein is obviously improved. The soluble expression level of the Q140L and S304G mutant proteins is slightly increased compared with that of the wild type.

(4) Determination of temperature stability of 2-methylbutyrate side chain hydrolase wild-type and mutant proteins

Setting the temperature conditions of the reaction tank of the PCR instrument, taking out the same amount of wild type and mutant proteins, placing the wild type and mutant proteins in the reaction tank with different temperatures (20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃,42 ℃, 45 ℃ and 50 ℃), incubating for 10 minutes, immediately placing the wild type and mutant proteins on ice to cool for 10 minutes, and taking out the proteins from the wild type and mutant proteins to perform enzyme activity determination, wherein the determination conditions are as follows: the concentration of the enzyme was 0.05. mu.M, the concentration of the substrate lovastatin was 400. mu.M, the reaction was carried out at 37 ℃ for 10 minutes, and the produced monacolin J was measured by HPLC. And calculating the relative value of the enzyme activity of the protein after different temperature treatments by taking the activity of the protein without temperature treatment as 100 percent, and drawing a curve. As shown in FIG. 9, T of mutants D106G, Q140L, S304G, D106G/Q140L, D106G/Q140L/S304G₅₀The values are respectively improved by 4 ℃, 3 ℃, 2 ℃, 5.5 ℃ and 8 ℃ compared with the wild type.

3. Construction of expression vector pRSFDuet-PcEST-M carrying gene encoding PcEST mutant D106G/Q140L/S304G

Mutation PCR: site-directed mutagenesis was performed using the QuikChange method. pRSFDuet-PcEST plasmid DNA is used as a template, primers (Table 1) are designed and synthesized according to mutation sites, and PCR amplification is carried out to obtain a coding sequence for coding a PcEST mutant D106G/Q140L/S304G.

The PCR reaction system constructed by the single-point mutant D106G comprises 32.5 mu L of sterile water, 1 mu L of template DNA and 5 XPrimeSTAR HS buffer (Mg²⁺plus), 4. mu.L of dNTP mixture (2.5 mmol/L each), 1. mu.L of forward primer D106G (20 pmol/. mu.L), 1. mu.L of reverse primer D106G (20 pmol/. mu.L), 0.5. mu.L of TaKaRa PrimeSTAR HS polymerase. The reaction conditions were pre-denaturation at 95 ℃ for 5min, at 94 ℃ for 10s, at 60 ℃ for 15s, at 72 ℃ for 7.5min,18 cycles, and final extension at 72 ℃ for 10 min.

Enzyme digestion: the PCR product after the reaction was taken out, the Dpn I enzyme was added in a ratio of 1:50(Dpn I enzyme: PCR product, v: v), and the mixture was digested at 37 ℃ for 1 hour.

The cells were removed, transformed into competent cells of Escherichia coli BL21(DE3), plated on LB plates containing kanamycin (final concentration: 50. mu.g/ml), and cultured overnight at 37 ℃.

After transformants grew, single clones were picked for sequencing.

The double-point mutant D106G/Q140L is constructed by carrying out PCR reaction on the basis of the single-point mutant D106G by using a primer Q140L in the forward direction and a primer Q140L in the reverse direction (Table 1) and introducing a mutation site of Q140L.

The three-point mutant D106G/Q140L/S304G is constructed by carrying out PCR reaction on the basis of the double-point mutant D106G/Q140L by using a primer S304G in the forward direction and a primer S304G in the reverse direction (Table 1) and introducing a mutation site of S304G.

The expression vector pRSFDuet-PcEST-M carrying the coding gene of the PcEST mutant D106G/Q140L/S304G is finally obtained through three times of mutation, the nucleotide sequence is shown as SEQ ID NO:18 in the sequence table, and the total length is 1200 bp. The sequencing results were consistent with expectations.

4. Construction of recombinant Escherichia coli simultaneously containing coding gene of PcEST mutant D106G/Q140L/S304G and coding gene of LovD9

The pRSFDuet-pcest-M and pET22b-lovd vectors were co-transformed into competent cells of E.coli expression strain BL21(DE3) at a molar ratio of 1: 1. Colonies grown on LB plates containing both ampicillin (final concentration of 100. mu.g/ml) and kanamycin (final concentration of 50. mu.g/ml) were picked, transformants were sequenced, and correct strains and the corresponding vectors pRSFDuet-PcEST-M and pET22b-LovD contained therein, i.e., recombinant E.coli carrying genes encoding PcEST mutants D106G/Q140L/S304G and LovD9, were selected as the nucleotide sequences of PcEST mutants D106G/Q140L/S304G (shown as SEQ ID NO:18) and LovD9 (shown as SEQ ID NO: 11), and designated as PLM3 strain.

Expression of PcEST mutant D106G/Q140L/S304G and LovD9 proteins in PLM3 Strain

The PLM3 strain was cultured in 100ml at 37 ℃ to the logarithmic growth phase, followed by addition of IPTG (final concentration of 0.1mM) as an inducer and culture at 25 ℃ for 16 hours to induce protein expression. The cells were collected, suspended in 100ml of Tris-HCl buffer (50mM, pH8.0), sonicated and centrifuged, the supernatant and the pellet were separated, and the pellet was suspended in 100ml of Tris-HCl buffer (50mM, pH 8.0). 10. mu.l of the supernatant and the pellet were subjected to SDS-PAGE protein electrophoresis analysis, and as a result, as shown in FIG. 3, both proteins of PcEST mutant D106G/Q140L/S304G and LovD9 were expressed in the cells in PLM3 strain. By comparing the expression of the proteins in PL1 and PLM3 strains, it can be seen that the soluble expression level of the PcEST wild type expressed in PL1 strain is lower, while the soluble expression level of PcEST mutant D106G/Q140L/S304G expressed in PLM3 strain is higher, indicating that the protein can be folded correctly when PcEST mutant D106G/Q140L/S304G is expressed at 25 ℃.

Determination of the enzymatic Activity of PcEST mutant and LovD9 in PLM3 Strain

The PLM3 strain was cultured in 100ml at 37 ℃ to the logarithmic growth phase, followed by addition of IPTG (final concentration of 0.1mM) as an inducer and culture at 25 ℃ for 16 hours to induce protein expression. The cells were collected and suspended in 100ml of Tris-HCl buffer (50mM, pH 8.0). The detection method of the PcEST enzyme activity is as described in example 1, the enzyme activity of the PcEST obtained through final calculation is 26.08 +/-3.45U/ml (the unit U of the enzyme activity is defined as the enzyme amount required for catalyzing and generating 1 mu M monacolin J per minute), and compared with the PcEST wild type, the PcEST mutant D106G/Q140L/S304G enzyme activity is obviously improved, because the soluble expression amount of the PcEST mutant D106G/Q140L/S304G protein is obviously improved (as shown in figure 3). The detection method of the LovD9 enzyme activity is as described in example 1, and the final calculation result shows that the enzyme activity of LovD9 is 9.82 +/-0.35U/ml (the unit of the enzyme activity is defined as the amount of enzyme required for catalyzing and generating 1 mu M simvastatin per minute).

7. Application of escherichia coli recombinant strain PLM3 in synthesis of simvastatin by whole-cell biocatalytic conversion of lovastatin

A powder of PLM3 strain was prepared and the procedure was as described in example 2. Simvastatin is synthesized by utilizing PLM3 bacterial powder, and the specific operation process is described in example 2. As can be seen from FIGS. 4 and 5, as the cell concentration increased, the time taken until the conversion rate reached the maximum decreased, specifically: when the cell concentrations were 3.13mg/ml, 4.38mg/ml and 6.25mg/ml, respectively, the time taken for the conversion to reach the maximum was 1 hour, 3 hours and 5 hours, respectively, and the conversion was 92%.

Compared with the PL1 strain, the conversion rate of the PLM3 strain is improved by nearly 1 time, because the soluble expression quantity, the enzyme activity and the thermal stability of the PcEST mutant D106G/Q140L/S304G in the PLM3 strain are obviously improved, so that the activity of the PcEST in a whole-cell catalyst is enhanced, sufficient substrates are provided for the acylation reaction of the LovD in the second step, and the total conversion rate is greatly improved finally.

Sequence listing

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

Zhejiang Haizheng pharmaceutical Co Ltd

<120> recombinant strains, methods and related enzymes for synthesizing simvastatin

<130> DP1F180073ZX

<160> 18

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Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Asp Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Gln Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

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Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

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Met Gly Ser Asn Ile Asp Ala Ala Val Ala Ala Asp Pro Val Val Leu

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Met Glu Thr Ala Phe Arg Lys Ala Val Glu Ser Ser Gln Ile Pro Gly

20 25 30

Ala Val Leu Met Ala Arg Asp Ala Ser Gly Arg Leu Asn Tyr Thr Arg

35 40 45

Cys Phe Gly Ala Arg Thr Val Arg Arg Asp Glu Asn Asn Gln Leu Pro

50 55 60

Pro Leu Gln Val Asp Thr Pro Cys Arg Leu Ala Ser Ala Thr Lys Leu

65 70 75 80

Leu Thr Thr Ile Met Ala Leu Gln Cys Met Glu Arg Gly Leu Val Arg

85 90 95

Leu Asp Glu Thr Val Asp Arg Leu Leu Pro Asp Leu Cys Ala Met Pro

100 105 110

Val Leu Glu Gly Phe Asp Asp Ala Gly Asn Pro Arg Leu Arg Glu Arg

115 120 125

Arg Gly Lys Ile Thr Leu Arg His Leu Leu Thr His Thr Ser Gly Leu

130 135 140

Ser Tyr Val Phe Leu His Pro Leu Leu Arg Glu Tyr Val Ala Gln Gly

145 150 155 160

His Leu Gln Gly Ala Glu Lys Phe Gly Ile Gln Asn Arg Phe Ala Pro

165 170 175

Pro Leu Val Asn Asp Pro Gly Ala Glu Trp Ile Tyr Gly Ala Gly Ile

180 185 190

Asp Trp Ala Gly Lys Leu Val Glu Arg Ala Thr Gly Leu Asp Leu Glu

195 200 205

Gln Tyr Leu Gln Glu Asn Ile Cys Ala Pro Leu Gly Ile Thr Asp Met

210 215 220

Thr Phe Lys Leu Gln Gln Arg Pro Asp Met Leu Ala Arg Arg Ala Asp

225 230 235 240

Met Thr His Arg Asn Ser Ser Asp Gly Lys Leu Arg Tyr Asp Asp Thr

245 250 255

Val Tyr Phe Arg His Asp Gly Glu Glu Cys Phe Gly Gly Gln Gly Val

260 265 270

Phe Ser Ser Pro Gly Ser Tyr Met Lys Val Leu His Ser Leu Leu Lys

275 280 285

Arg Asp Gly Leu Leu Leu Gln Pro Gly Thr Val Asp Leu Met Phe Gln

290 295 300

Pro Ala Leu Glu Pro Arg Leu Glu Glu Gln Met Asn Gln His Met Asp

305 310 315 320

Ala Ser Pro His Ile Asn Tyr Gly Gly Pro Met Pro Met Val Met Arg

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Arg Ser Phe Gly Leu Gly Gly Ile Ile Ala Leu Glu Asp Leu Asp Gly

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Glu Asn Trp Arg Arg Lys Gly Ser Met Thr Phe Gly Gly Gly Pro Asn

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Ile Ile Trp Gln Ile Asp Pro Lys Ala Gly Leu Cys Thr Leu Val Phe

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Phe Gln Leu Glu Pro Trp Ser Asp Pro Val Cys Arg Asp Leu Thr Arg

385 390 395 400

Thr Phe Glu Lys Ala Ile Tyr Ala Gln Tyr Gln Gln Gly

405 410

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<213> Artificial Sequence (Artificial Sequence)

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Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Gly Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Gln Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

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<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Asp Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Leu Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 5

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Asp Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Gln Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Gly

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 6

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Gly Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Leu Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Ser

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 7

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Gly Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Gln Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Gly

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 8

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Asp Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Leu Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Gly

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 9

<211> 399

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Asp Thr Thr Phe Gln Ala Ala Ile Asp Thr Gly Lys Ile Asn Gly

1 5 10 15

Ala Val Val Cys Ala Thr Asp Ala Gln Gly His Phe Val Tyr Asn Lys

20 25 30

Ala Thr Gly Glu Arg Thr Leu Leu Ser Gly Glu Lys Gln Pro Gln Gln

35 40 45

Leu Asp Asp Val Leu Tyr Leu Ala Ser Ala Thr Lys Leu Ile Thr Thr

50 55 60

Ile Ala Ala Leu Gln Cys Val Glu Asp Gly Leu Leu Ser Leu Asp Gly

65 70 75 80

Asp Leu Ser Ser Ile Ala Pro Glu Leu Ala Ala Lys Tyr Val Leu Thr

85 90 95

Gly Phe Thr Asp Asp Glu Ser Pro Leu Gly Asp Pro Pro Ala Arg Pro

100 105 110

Ile Thr Leu Lys Met Leu Leu Thr His Ser Ser Gly Thr Ser Tyr His

115 120 125

Phe Leu Asp Pro Ser Ile Ala Lys Trp Arg Ala Leu Tyr Ala Asn Pro

130 135 140

Glu Asn Glu Lys Pro Arg Leu Val Glu Glu Met Phe Thr Tyr Pro Leu

145 150 155 160

Ser Phe Gln Pro Gly Thr Gly Trp Met Tyr Gly Pro Gly Leu Asp Trp

165 170 175

Ala Gly Arg Val Val Glu Arg Val Thr Gly Gly Thr Leu Met Glu Phe

180 185 190

Met Gln Lys Arg Ile Phe Asp Pro Leu Gly Ile Thr Asp Ser Gln Phe

195 200 205

Tyr Pro Val Thr Arg Glu Asp Leu Arg Ala Arg Leu Val Asp Leu Asn

210 215 220

Pro Ser Asp Pro Gly Ala Leu Gly Ser Ala Val Ile Gly Gly Gly Gly

225 230 235 240

Glu Met Asn Leu Arg Gly Arg Gly Ala Phe Gly Gly His Gly Leu Phe

245 250 255

Leu Thr Gly Leu Asp Phe Val Lys Ile Leu Arg Ser Leu Leu Ala Asn

260 265 270

Asp Gly Met Leu Leu Lys Pro Ala Ala Val Asp Asn Met Phe Gln Gln

275 280 285

His Leu Gly Pro Glu Ala Ala Ala Ser His Arg Ala Ala Leu Ala Gly

290 295 300

Pro Leu Gly Pro Phe Phe Arg Val Gly Thr Asp Pro Glu Thr Lys Val

305 310 315 320

Gly Tyr Gly Leu Gly Gly Leu Leu Thr Leu Glu Asp Val Asp Gly Trp

325 330 335

Tyr Gly Glu Arg Thr Leu Thr Trp Gly Gly Gly Leu Thr Leu Thr Trp

340 345 350

Phe Ile Asp Arg Lys Asn Asn Leu Cys Gly Val Gly Ala Ile Gln Ala

355 360 365

Val Leu Pro Val Asp Gly Asp Leu Met Ala Asp Leu Lys Gln Thr Phe

370 375 380

Arg His Asp Ile Tyr Arg Lys Tyr Ser Ala Trp Lys Gly Gln Gln

385 390 395

<210> 10

<211> 1200

<212> DNA

<213> Penicillium chrysogenum (Penicillium chrysogenum)

<400> 10

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacaa 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 11

<211> 1239

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgggcagca acattgatgc ggctgtggcg gcggaccctg tggtgttaat ggaaaccgcg 60

tttcgcaaag cggtggaaag cagccaaatt ccgggcgcgg ttttaatggc gcgtgatgcg 120

tcaggtcgcc tgaattatac ccgctgcttt ggtgcacgta ccgttcgtcg cgatgaaaac 180

aaccaactgc cgccgctgca agttgatacc ccgtgtcgtt tagcgagcgc gaccaaactg 240

ctgaccacca ttatggcgct gcaatgcatg gaacgtggcc tggtgcgttt agatgaaacc 300

gtggatcgcc tgttacctga tctgtgcgcg atgcctgttc tggaaggctt tgatgatgcg 360

ggtaacccgc gcttacgtga acgtcgtggc aaaattaccc tgcgccatct gttaacccat 420

accagcggcc tgagctatgt gtttctgcat ccgctgctgc gcgaatatgt tgcgcagggc 480

catctgcaag gcgcggaaaa atttggcatt cagaaccgct ttgcgccgcc gctggttaat 540

gatcctggcg cggaatggat ttatggcgcg ggcattgatt gggcgggcaa attagttgaa 600

cgcgcgaccg gcttggatct ggaacagtat ctgcaggaaa acatttgtgc gccgctgggc 660

attaccgata tgacctttaa actgcagcag cgcccggata tgttagcacg ccgtgcggat 720

atgacccatc gcaacagcag cgatggcaaa ctgcgctatg atgacaccgt gtattttcgc 780

catgatggcg aagaatgctt tggtggccag ggcgtgttta gctcaccggg cagctatatg 840

aaagtgctgc atagcctgct gaaacgcgat ggcctgctgt tacagcctgg taccgtggat 900

ctgatgtttc agccggcact ggaaccgcgt ttagaagaac agatgaacca gcacatggat 960

gcgagcccgc atattaatta tggcggcccg atgcctatgg ttatgcgccg cagctttggc 1020

ttaggcggca ttattgcgct ggaagacctg gatggcgaaa actggcgccg taaaggcagc 1080

atgacctttg gtggcggccc gaacattatt tggcagattg atccgaaagc gggcttatgt 1140

accctggtgt tttttcagct ggaaccgtgg tcagatcctg tgtgccgcga tttaacccgc 1200

acctttgaga aagcgattta tgcgcagtat cagcagggc 1239

<210> 12

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctgggcga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacaa 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgct ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 13

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacta 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 14

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacaa 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcag gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 15

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctgggcga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacta 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgct ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcaa gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 16

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctgggcga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacaa 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgct ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcag gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 17

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctggacga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacta 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcag gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

<210> 18

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggatacca cctttcaggc ggcgattgat accggcaaaa ttaacggcgc agttgtttgc 60

gcaaccgacg cacagggcca ttttgtttat aacaaagcaa ccggcgaacg taccctgctg 120

tctggcgaaa aacaaccgca acagctggat gatgttctgt atctggcaag cgcgaccaaa 180

ctgattacca ccattgctgc tctgcaatgc gttgaagacg gtctgctgag tctggacggc 240

gatctgagta gtattgcacc ggaactggca gcgaaatacg ttctgaccgg ttttaccgac 300

gacgaaagtc cgctgggcga tccgccggca cgtccgatta ccctgaaaat gctgctgacc 360

catagcagcg gtaccagcta tcatttcctg gatccgtcta tcgcaaaatg gcgcgcacta 420

tacgcgaatc cggaaaacga aaaaccgcgt ctggtcgaag agatgttcac ctatccgctg 480

agttttcaac cgggtaccgg ctggatgtac ggtccgggtc tggattgggc aggtcgcgtt 540

gttgaacgtg ttacgggcgg taccctgatg gaattcatgc agaaacgcat cttcgatccg 600

ctgggtatca ccgatagcca gttttatccg gttacccgcg aagatctgcg cgcacgtctg 660

gttgatctga atccgtctga tccgggcgca ctgggttctg cagttattgg cggcggcggt 720

gaaatgaatc tgcgcggtcg cggcgcattt ggcggtcacg gtctgtttct gaccggtctg 780

gatttcgtca aaatcctgcg tagcctgctg gctaacgacg gtatgctgct gaaaccggct 840

gctgtcgata acatgttcca gcagcatctg ggtccggaag cagcagcaag tcatcgcgca 900

gcactggcag gtccgctggg tccgtttttc cgcgttggta ccgatccgga aaccaaagtt 960

ggttacggtc tgggcggtct gctgaccctg gaagacgttg acggttggta cggcgaacgt 1020

accctgacct ggggcggtgg tctgaccctg acctggttta tcgaccgcaa aaacaacctg 1080

tgtggtgttg gcgcaattca agcagttctg ccggttgacg gcgatctgat ggcagatctg 1140

aaacagacct tccgccacga tatctaccgc aaatacagcg cctggaaagg tcagcagtga 1200

Claims

1. A2-methylbutyrate side chain hydrolase, which is:

(1) PcEST/D106G: the amino acid sequence is shown as SEQ ID NO. 3;

(2) PcEST/S304G: the amino acid sequence is shown as SEQ ID NO. 5;

(3) PcEST/D106G/Q140L: the amino acid sequence is shown as SEQ ID NO. 6; or

(4) PcEST/D106G/Q140L/S304G: the amino acid sequence is shown in SEQ ID NO. 9.

2. A biomaterial, being:

(1) a nucleic acid molecule encoding the 2-methylbutyrate side chain hydrolase of claim 1; or

3. The biomaterial of claim 2, wherein the nucleic acid molecule is PcEST/D106G: the nucleotide sequence is shown as SEQ ID NO:12, PcEST/S304G: the nucleotide sequence is shown as SEQ ID NO:14, PcEST/D106G/Q140L: the nucleotide sequence is shown as SEQ ID NO. 15 or PcEST/D106G/Q140L/S304G: the nucleotide sequence is shown in SEQ ID NO. 18.

4. Use of the 2-methylbutyrate side chain hydrolase according to claim 1 for hydrolyzing statins having a 2-methylbutyrate side chain.

5. The use according to claim 4, wherein the statin-like substance having a side chain of 2-methylbutyric acid is lovastatin, mevastatin or pravastatin.

6. The use according to claim 4 or 5, wherein the statin is in the acid, lactone or salt form.

7. A recombinant strain for synthesizing simvastatin, which expresses both 2-methylbutyrate side chain hydrolase according to claim 1 and acyltransferase.

8. The recombinant strain of claim 7, wherein the recombinant strain is E.

9. The recombinant strain of claim 8, wherein the recombinant strain is Escherichia coli BL21(DE3) strain.

10. The recombinant strain according to claim 7, wherein the amino acid sequence of said acyltransferase is represented as SEQ ID NO 2.

11. The recombinant strain according to any one of claims 7 to 10, wherein the recombinant strain carries a gene encoding a 2-methylbutyrate side chain hydrolase and a gene encoding an acyltransferase.

12. The recombinant strain according to claim 11, wherein the nucleotide sequence of the gene encoding the 2-methylbutyrate side chain hydrolase is represented by any one of SEQ ID NO 12, 14-15 or 18, and the nucleotide sequence of the gene encoding the acyltransferase is represented by SEQ ID NO 11.

13. A method of constructing a recombinant strain according to any one of claims 7 to 12, comprising the steps of: co-transforming Escherichia coli with a plasmid carrying the gene encoding 2-methylbutyrate side chain hydrolase according to claim 1 and a plasmid carrying the gene encoding acyltransferase, and screening to obtain a recombinant strain simultaneously expressing 2-methylbutyrate side chain hydrolase and acyltransferase.

14. The method according to claim 13, wherein the nucleotide sequence of the gene encoding 2-methylbutyrate side chain hydrolase is represented by any one of SEQ ID NO 12, 14-15 or 18, and the nucleotide sequence of the gene encoding acyltransferase is represented by SEQ ID NO 11.

15. Use of the recombinant strain of any one of claims 7 to 12 or a bacterial suspension thereof or a fermentation broth thereof or bacterial powder thereof in the synthesis of simvastatin.

16. A method for synthesizing simvastatin, which comprises using the recombinant strain of any one of claims 7-12, and using lovastatin and acyl thioester as substrates to catalyze the conversion of lovastatin into simvastatin.